Figure 4: Sphere culture of bovine peripheral corneal endothelial cells. (a) Floating spheres on day 7 in defined serum-free media. Aggregation and development of dark cores can occur when spheres are left over the optimal culturing period of 5–7 days. (b) Cells migrating from an attached sphere on adherent substrate. The arrowheads show the contour of the sphere. (c) Nestin (green: undifferentiated cell marker) and (d) β-III tubulin (red: neuronal marker) staining were detected in the cells that migrated from the primary spheres. Nuclei were counterstained with DAPI (blue). Insets are negative controls with nonimmunized IgG. (a–c) Scale bar = 100 μm; (d) Scale bar = 50 μm.